Background/Rationale: A limited number of persons infected with HIV-1 develop circulating plasma antibodies that potently neutralize a wide variety of HIV-1 isolates. The characteristics and specificities of such antibodies can guide the development of HIV-1 vaccine candidates. Current methods for the study of these antibodies include isolation of antibodies from memory B cells, which may not always reflect the circulating antibodies. Our research has focused on the identification and characterization of broad neutralizing antibodies directly from patient plasma (without potential bias of selection). We have noted particular biochemical signatures that point to a common dominant, acidic epitope that is targeted on the envelope of HIV. Objectives: The specific hypothesis of this proposal is that a shared acidic epitope on the gp120 envelope binds a shared immunoglobulin gene family giving rise to the broad HIV-1 neutralizing response in plasma. The specific aims of the project are to 1) Directly isolate and sequence the antibodies responsible for the broad neutralization from the plasma of HIV-1 infected individuals with broad neutralization, 2) Define the gene family usage of neutralizing versus non-neutralizing antibodies to test the hypothesis that a shared immunoglobulin gene family is responsible for the broad HIV-1 neutralization response in plasma, 3) Map the corresponding epitope(s) of the broad neutralizing antibodies to test the hypothesis that a shared acidic epitope of gp120 is responsible for the broad HIV-1 neutralization response in plasma. Methods: We have identified 10 patients with broad neutralization, of which 3 will be studied in detail. The affinity purification (antigen, subclass, and light chain specific) and fractionation (free-flow isoelectric focusing) scheme can narrow the antibodies of interest, directly, from the plasma to individual species, which will be tested for broad neutralization. Upon confirmation, the individual antibody bands will be sequenced de novo using LC-MS. Epitope mapping of the new mAbs will be undertaken with Elisa, mutagenesis studies, and X-ray crystallography. Once the epitope is identified, Elisa and mutagenesis studies will be used to test the active fraction o the other 7 individuals to determine if this epitope is responsible for broad neutralization. We wil also define the gene family usage of neutralizing versus non-neutralizing antibodies in 20 patients. Anticipated results will be isolation of new mAbs and the identification of a common acidic epitope that can direct broad HIV-1 neutralization. We have completed enough work on the techniques described herein (as well as each alternative plan) to ensure that the aims are feasible and will be completed. Impact: If our hypothesis proves correct, then the results will be novel in its method, and directl applicable to the study of HIV vaccines. This study will provide a deeper understanding of possibilities of the broad HIV-1 neutralizing response in humans, and it will also have identified a naturally occurring epitope(s) that can be the target of potent cross-clade antibodies against HIV-1 as well as the gene family(ies) responsible.